Methodology was developed to study the activity of deoxycytidylate deaminase and thymidylate synthetase in intact cells and to study the flux through the de novo pyrimidine pathway in intact cells and in various tissues in vivo. The dynamics of thymidylate synthetase inhibition by MTX and fluoropyrimidines and the effects of altered nucleotide pools were investigated in intact L1210 cells. The role of 3-deazauridine as a fraudulent allosteric regulator of carbamyl phosphate synthetase II was demonstrated. Several compounds to inhibit uridine kinase were synthesized and evaluated. FUdR-5'-methylphosphonate, an analogue of the activated form of FUdR, was synthesized and evaluated. Acivicin, PALA, pyrazofurin, and 6-azauridine were evaluated for inhibition of de novo pyrimidine biosynthesis in normal and tumorous tissues in vivo using precursor molecules (glutamine, ammonium, bicarbonate) labeled with stable isotopes of carbon and nitrogen and quantitation by GC/MS. The salvage of CdR by L1210 cells and incorporation into cytosine and thymine bases of DNA was studied. The activation of mAMSA by microsomal and non-microsomal enzymes and the role of activation in cytotoxicity was investigated.